WO2017033922A1 - Method of detecting target substance using field-effect transistor - Google Patents

Abstract

[Problem] To provide a novel method for detecting the presence or absence of a target substance in a test sample, using a field-effect transistor. [Solution] Provided is a method characterized by including: (a) a step in which a complex including magnetic particles and a factor which binds specifically to the target substance is brought into contact with a test sample in a solvent; (b) a step in which, after step (a), the solvent is removed and the complex is dispersed in water having a specific resistance of at least 1 MΩ·cm; (c) a step in which, after step (b), the electrical characteristics of the complex are measured using the field-effect transistor while a magnetic force is used to bring the complex into close contact with the surface of the gate electrode of the field-effect transistor; and (d) a step in which the presence or absence of the target substance in the test sample is detected by comparing the electrical characteristics measured in (c) with electrical characteristics obtained in a control experiment in which the complex is not brought into contact with the target substance.

Description

Method for detecting target substance using field effect transistor

The present invention relates to a method for detecting the presence / absence of a target substance in a test sample using a field effect transistor, and an apparatus for carrying out the method.

A field effect transistor (Field Effect Transistor, FET) is an n-type semiconductor drain and source formed on a p-type semiconductor, and a p-type semiconductor channel is insulated by silicon oxide to form a gate. An electron depletion layer is generated in the channel by the potential applied to the channel, and the current flowing between the drain and the source can be changed.

Although research has been conducted on FET sensors that detect antigen-antibody reactions, no satisfactory results have been obtained. An FET sensor (Immuno-FET) for detecting an antigen-antibody reaction that has been studied so far immobilizes an antibody or antigen on the surface of a gate insulating film, and when an antigen-antibody complex is formed, the charge on the surface of the gate insulating film is This was based on the concept of changing the electrical conductivity between the source and the drain and detecting the change with the FET. However, since the typical size of an antibody is about 10 nm, while the Debye length on the surface of the gate insulating film in a physiological salt solution is about 1 nm, when the antibody is immobilized on the surface of the gate insulating film, The antigen in the solution was bound to the antibody outside the Debye length, and the charge of the antigen was shielded by the counter ion, making it difficult in principle to be detected by the FET (see also FIG. 1 of this application). thing).

As a method for solving the above-mentioned problems, the present inventors use an antibody divided into a VH region and a VL region, and a complex comprising a peptide containing the VH region, a peptide containing the VL region, and a target substance. Proposes a method of detecting an antigen / antibody complex directly by using a field effect transistor without using a secondary antibody by forming the complex and measuring the complex with an electrolytic effect transistor sensor. (Patent Document 1).

JP 2009-133800 A

The invention described in the above-mentioned Patent Document 1 is an epoch-making one in that the FET sensor that detects an antigen-antibody reaction, which has been considered difficult to realize, has been developed for the first time in the world, but it detects a target substance. Therefore, it was necessary to prepare an antigen-binding fragment of an antibody corresponding to the target substance. In addition, depending on the position of the epitope of the target substance, the charge change that the device should capture may occur outside the Debye length due to the three-dimensional structure of the protein. In such cases, stable measurement can be performed. could not.

In order to further increase the availability of an FET sensor for detecting an antigen-antibody reaction, the present inventors use an existing antibody (for example, a commercially available monoclonal antibody for a target substance) to detect an antigen-antibody reaction. Worked on sensor development. As a result, the present inventors created a complex in which an antibody that specifically binds to the target substance was bound to the surface of the magnetic particle, and adhered the complex to the surface of the gate electrode by magnetic force. The antigen-antibody reaction of the full-length antibody was successfully detected (see also the image diagram of FIG. 1 of the present application).

In this specification, a complex in which a factor that specifically binds to a target substance is bonded to the surface of a magnetic particle is created, and the complex is adhered to the surface of the gate electrode by magnetic force. The method for detecting the binding with the target substance may be referred to as the Molecular Charge Contact method (MCC method) .

In addition, since the FET sensor detects a change in the electrical characteristics of the object, measurement is performed in a state where the object exists in a buffer solution (having a property of conducting electricity) according to a conventional method. However, in the course of the experiment, the present inventors may cause various ions contained in the buffer solution to decrease the detection sensitivity of the change in the electrical characteristics of the object using the FET sensor, In other words, I noticed that various ions contained in the buffer paired with the charge of the antigen to be detected outside the Debye length, resulting in a decrease in detection sensitivity.

The complex of magnetic particles and antibodies used in the method of the present invention itself has the property of becoming charged particles in water and conducting electricity through the aqueous solution. Therefore, the inventors of the present invention performed a measurement in a low ion concentration state in which only the measurement object exists in pure water in the method of the present invention. Surprisingly, the object was in the buffer solution. Compared with the case where it was measured in the existing state, the detection limit concentration of the target substance was drastically decreased, and the detection sensitivity was increased several tens of times (see Examples of the present application). From these findings, the present invention has been completed.

That is, the present invention, in one embodiment, is a method for detecting the presence / absence of a target substance in a test sample using a field effect transistor,
(A) contacting a test sample with a complex containing a factor that specifically binds to the target substance and a magnetic particle in a solvent;
(B) after step (a), removing the solvent and dispersing the composite in water having a specific resistance of 1 MΩ · cm or more;
(C) after step (b), measuring the electrical characteristics of the composite with the field effect transistor while the composite is brought into close contact with the gate electrode surface of the field effect transistor by magnetic force; and
(D) The presence or absence of the target substance in the test sample by comparing the electrical characteristics measured in step (c) with those in a control experiment in which the complex is not contacted with the target substance. Detecting the absence,
It is related with the method characterized by including.

In one embodiment of the present invention, the “water having a specific resistance of 1 MΩ · cm or more” is water having a specific resistance of 15 MΩ · cm or more.

In one embodiment of the present invention, the “factor that specifically binds to the target substance” specifically binds to the antibody or antigen-binding fragment thereof that specifically binds to the target substance, or to the target substance. It is a nucleic acid.

In one embodiment of the present invention, the target substance is biotin, and the “factor that specifically binds to the target substance” is streptavidin.

In one embodiment of the present invention, the gate surface of the field effect transistor is surface-treated so as to generate a surface functional group in a solution.

In one embodiment of the present invention, the surface of the gate electrode of the field effect transistor is coated with an oxide or a nitride.

In one embodiment of the present invention, the oxide or nitride is tantalum oxide, aluminum oxide, silicon nitride, titanium oxide, or silicon oxide.

In one embodiment of the present invention, when the surface of the gate electrode of the field effect transistor is coated with a metal that does not substantially generate a surface functional group in the solution, the surface functionality is applied to the surface of the gate electrode in the solution. It is further characterized in that it is surface-treated so as to form a group.

In one embodiment of the present invention, the metal is gold or platinum.

In one embodiment of the present invention, the field effect transistor is an Ion Sensitive FET.

Another embodiment of the present invention is an apparatus for detecting the presence / absence of a target substance in a test sample,
Field effect transistors,
A complex comprising a factor that specifically binds to the target substance and magnetic particles, and
A magnetic force generation source for drawing the composite to the surface of the gate electrode of the field effect transistor;
The apparatus measures the electrical characteristics of the composite while bringing the composite into close contact with the surface of the gate electrode of the field effect transistor by magnetic force, and the measured electrical characteristics are contacted with the target substance. By detecting the presence / absence of the target substance in the test sample by comparing with the electrical characteristics in the control experiment when not
The measurement relates to an apparatus in which the complex is performed in a state where the complex exists in water having a specific resistance of 1 MΩ · cm or more.

The invention described above arbitrarily combining one or more features of the present invention is also included in the scope of the present invention.

FIG. 1 is a schematic diagram of an immuno-FET that is conventionally assumed and an immuno-FET according to an embodiment of the present invention. FIG. 2 shows a schematic diagram of an experimental outline of the MCC method using a biotin-streptavidin system, which is an embodiment of the present invention. FIG. 3 shows the experimental results of the MCC method (measured in pure water) using the biotin-streptavidin system, which is an embodiment of the present invention. FIG. 4 shows the experimental results of the MCC method (measured in a buffer) using a biotin-streptavidin system. FIG. 5 shows a schematic diagram of an antigen-antibody reaction on magnetic particles and an experimental result of an MCC method using an antigen-antibody reaction system, which is an embodiment of the present invention.

In the method of the present invention, the presence / absence of a target substance in a test sample is detected using a field effect transistor (FET). A field effect transistor has a drain and a source formed of an n-type semiconductor on a p-type semiconductor, and a gate formed by insulating the channel of the p-type semiconductor with silicon oxide. A metal oxide semiconductor (Metal Oxide Semiconductor, It is a kind of transistor having a (MOS) structure. In an FET, an electron depletion layer is generated in a channel by a potential applied to a gate, and a current flowing between a drain and a source can be changed. The field effect transistor used in the present invention may be, for example, an Ion Sensitive Field Effect Transistor (ISFET).

The method of the present invention includes a step of contacting a test sample with a complex containing a factor that specifically binds to a target substance and a magnetic particle in a solvent. In this step, the type of solvent is not limited and can be appropriately selected by those skilled in the art depending on the type of binding reaction. For example, the solvent used in this step may be a buffer solution, or pure water or ultrapure water. Also, the reaction conditions between the complex and the target substance can be appropriately selected by those skilled in the art depending on the nature of each factor.

In the present invention, the combination of “target substance” and “factor that specifically binds to target substance” is a combination of two types of substances / factors that specifically bind (or a combination of two or more types of substances / factors). It is possible to use various substance / factor combinations known so far. Further, a combination of a certain “target substance” and “factor that specifically binds to the target substance” used in the present invention can be applied to the present invention even if the combination is reversed (for example, “ When “target substance” is “A” and “factor that specifically binds to target substance” is “B”, “target substance” is “B” and “factor that specifically binds to target substance” is “ A "can also be used).

For example, “an agent that specifically binds to a target substance” includes an antibody that specifically binds to the target substance (for example, a monoclonal antibody that specifically binds to the target substance) or an antibody-binding fragment thereof, or a target substance. A nucleic acid that specifically binds (for example, a nucleic acid having a sequence complementary to the target nucleic acid) can be used. Specific interactions between known proteins (eg, biotin-streptavidin combinations, receptor-ligand (or agonist) combinations, enzyme-substrate combinations), specific molecules and specific Binding aptamers (for example, nucleic acid aptamers, peptide aptamers), specific interactions between compounds (for example, glucose and phenylboronic acid), molecular templates manufactured to specifically bind to specific molecules Polymers and the like can also be applied to the present invention.

Various materials can be used for the magnetic particles used in the present invention. For example, magnetite (Fe ₃ O ₄ ), various ferrites (XFe ₂ O ₄ , X = Mn, Co, Ni, Mg, Cu) , Li _0.5 , Fe _0.5, etc.), ferric trioxide (Fe ₂ O ₃ ), etc.

The “complex containing a factor that specifically binds to a target substance and a magnetic particle” used in the present invention is obtained by directly or indirectly binding a “factor that specifically binds to a target substance” and a magnetic particle. It may be a thing. The mode of binding between the “factor that specifically binds to the target substance” and the magnetic particles is not particularly limited. Depending on the nature of the “factor that specifically binds to the target substance” and the nature of the magnetic particles to be used, May be combined in any suitable manner. For example, as performed in the examples of the present application, the “factor that specifically binds to the target substance” is formed on the surface of the magnetic particles via a spacer (carboxylic acid group-modified magnetic particles and diaminodecane are used in the examples of the present application). For example, a magnetic particle directly or indirectly bound with streptavidin is prepared, and a biotinylated “factor that specifically binds to a target substance” (for example, a biotin-labeled antibody) is prepared. According to the binding method, a “complex including a factor that specifically binds to a target substance and magnetic particles” used in the present invention can be prepared.

In the method of the present invention, the “target substance” and the “target substance” are brought into close contact with the gate surface of the field effect transistor by a magnetic force so that the “complex containing a factor that specifically binds to the target substance and the magnetic particles” is adhered to the gate surface of the field effect transistor. It is possible to measure a change in electrical characteristics caused by the binding with a factor that specifically binds to the ". The generation method of the “magnetic force” used in the present invention is not limited, and for example, a magnet or an electromagnet can be used.

In the present invention, measurement is performed in a state where the “composite”, which is a measurement target of the electrical characteristics by the field effect transistor, exists in water having a specific resistance of 1 MΩ · cm or more. In the present specification, water having a specific resistance of 1 MΩ · cm or higher is sometimes referred to as “pure water”, and water having a specific resistance of 15 MΩ · cm or higher is referred to as “ultra pure water”. “Pure water” and “ultra pure water” used in the present invention may be those commercially available for experiments, using water produced using a commercially available pure water production apparatus or ultra pure water production apparatus. May be. The principle of water purification of the apparatus for producing “pure water” or “ultra pure water” used in the present invention is not limited. For example, water is purified by an ion exchange resin, a reverse osmosis membrane, or a combination thereof. It may be.

In one embodiment of the present invention, an Ion Sensitive FET is used. In the Ion Sensitive FET, an ion layer needs to be formed on the gate electrode surface as a premise of the measurement principle (for example, when the gate surface is coated with tantalum oxide, the gate surface is a solution (water, buffer, etc.) When a contact is made, a hydroxyl group (that is, a surface functional group) is formed on the coated surface, and the hydroxyl group captures a hydrogen ion in the solution to form a layer of ions). When a substance (having charged particles) comes into contact with a layer of ions formed on the surface of the gate electrode, an electrical change occurs on the surface of the gate electrode. The electrical change that occurs on the surface of the gate electrode varies depending on the electrical state of the substance in contact (for example, the presence or absence of binding to other substances), so the value measured on the measurement object and the measurement object By comparing the value measured before the change in electrical state (ie, control), the presence or absence of a change in the electrical state of the substance can be detected.

That is, the surface of the gate electrode of the field effect transistor used in the method of the present invention may be processed as long as it can be used in the method of the present invention. The surface of the gate electrode surface-treated so as to generate a surface functional group in a solution (for example, in water or in a buffer solution) can be suitably used. Examples of such surface treatments include surface coatings with oxides or nitrides (more specifically, metal oxides or metal nitrides), more preferably tantalum oxide, aluminum oxide, nitridation. Surface coating with silicon, titanium oxide, or silicon oxide can be used.

In addition, among metals generally used as gate surfaces of field effect transistors, even when a metal that does not substantially generate surface functional groups in solution (for example, gold, platinum, etc.) is used, Used in the present invention by further modifying the surface with a functional group such as a group, or by further coating a substance that generates a surface functional group in a solution (for example, an organic substance such as an oxide, nitride, or polymer). be able to. The method for modifying the functional group on the metal surface is not particularly limited, and can be appropriately performed by those skilled in the art using known methods.

The terms used in this specification are used to describe specific embodiments except for those specifically defined, and are not intended to limit the invention.

In addition, the term “comprising” as used herein is intended to mean that there is a matter (member, step, element, number, etc.) described, unless the context clearly requires a different understanding. It does not exclude the presence of other items (members, steps, elements, numbers, etc.).

Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, unless otherwise defined, idealized, or overly formal. It should not be interpreted in a general sense.

In the following, the present invention will be described in more detail with reference to examples. However, the invention can be embodied in various ways and should not be construed as limited to the embodiments set forth herein.

Example 1: MCC method using biotin-streptavidin system (measured in ultrapure water)

The inventors first conducted an experiment using the MCC method using a biotin-streptavidin system as a model of the MCC method using an antigen-antibody reaction system.

(1) Modification of the surface of magnetic beads with amino groups In this example, φ1 μm magnetic beads (7-12 × 10 ⁹ beads / ml, Dynabeads MyOne Carboxylic Acid, purchased from Invitrogen) having carboxylic acid groups on the surface were used. used. A 50 μl solution containing magnetic beads was dispersed in a microtube and washed twice with deionized water. The supernatant was removed while attracting the magnetic beads using a magnet. Diaminodecane (1,10-diaminothecode) in heated deionized water, containing 6 mM WSC (1-ethyl-3- (3-dimethyl-aminopropylol) carbodiimide hydrochloride, Dojindo Laboratories Co., Ltd.) , Ltd.) was dissolved at a saturated concentration to prepare a diaminodecane solution. The magnetic beads were dissolved in 100 ul of the diaminodecane solution and left at 50 ° C. overnight to modify the amino group on the surface of the magnetic beads. Then, the magnetic beads were collected by a magnet and washed with deionized water twice at room temperature to remove residual reagents.

The magnetic beads at this stage have one of the two amino groups of diaminodecane bonded to the carboxylic acid group on the surface of the magnetic beads, and the other amino group not used for bonding is free. .

(2) Immobilization of biotin on magnetic beads By dissolving biotin ((+)-Biotin, Wako Co., Ltd.) in deionized water containing 60 mM WSC, a biotin solution with a concentration of 0.9 mM was obtained. Got ready. The magnetic beads prepared in (1) were dissolved in 500 μl of the above biotin solution and allowed to stand overnight at 50 ° C. to immobilize biotin on the amino groups on the surface of the magnetic beads. Then, the magnetic beads were collected by a magnet, and washed with deionized water twice at room temperature, thereby removing unimmobilized biotin.

(3) Biotin-streptavidin reaction on magnetic beads A biotin-streptavidin reaction was performed on the biotinylated surface of the magnetic beads. 1437 μl of phosphate buffered saline (PBS pH 7.4 (1 ×), gibco) containing 52.2 ug / ml of streptavidin (Streptavidin, Wako Co., Ltd.) Dissolved in. Then, PBS containing magnetic beads having a biotinylated surface was incubated at room temperature for 30 minutes to react biotin with streptavidin. After the reaction, the magnetic beads were attracted by a magnet and washed with PBS and deionized water at room temperature.

In this example, the biotinylated magnetic bead-streptavidin complex prepared here (sometimes referred to as sample magnetic bead in this example) is the “factor that specifically binds to the target substance”. And a composite containing magnetic particles ”. Streptavidin forms a tetramer and has the ability to bind to 4 molecules of biotin, so it has the ability to bind to additional biotin after binding to biotinylated magnetic beads (FIG. 2). checking).

(4) Reaction of biotinylated magnetic bead-streptavidin complex with biotin The biotinylated magnetic bead-streptavidin complex (sample magnetic bead) prepared in (3) is dispersed with 50 μl of PBS (-). The concentration was adjusted to a stock solution concentration (7-12 × 10 ⁹ beads / ml) and dispensed into a microtube so that 4 μl of one sample was obtained. Biotin to be reacted was prepared using PBS (−) at concentrations of 0.9 mM, 0.9 μM, 0.9 nM, 0.9 pM, 0.9 fM, 90 aM, 9 aM, and 0.9 aM, respectively.

Remove the supernatant of the dispensed sample magnetic beads while pulling the sample magnetic beads with a magnet, and then add 115 μl of biotin PBS (−) solution at each concentration to the microtube containing 4 μl of sample magnetic beads. After stirring by pipetting, the mixture was reacted at room temperature for 30 minutes. After the reaction, while pulling the sample magnetic beads with a magnet, the sample magnetic beads were washed twice with PBS (−) at room temperature, and unreacted biotin was washed away. After washing, the sample magnetic beads were immersed in ultrapure water to prevent drying until used for measurement.

(5) Measurement of Biotin-Streptavidin Reaction by Molecular Charge Contact (MCC) Method For measurement of electric signal by MCC method, a commercially available N-channel depletion type Ion Sensitive FET chip (ISFET COM Co., Ltd.) that can confirm the Nernst response. .) Was used. As the gate structure of the FET chip, the insulating film has a laminated structure of Ta ₂ O ₅ / SiO ₂ , and the film thickness is about 120 nm in total. At the time of measurement, a glass ring (inner diameter: 10 mm, thickness: 1 mm) is attached with an epoxy resin so that the gate sensing part (10 × 340 μm) of the FET can be immersed in 400 μl of the measuring solution so that the sample magnetic particles are attracted to the sensing part. A magnet (neodymium, approximately 26 × 76 mm) was laid under the ISFET. The FET electrical characteristics such as gate voltage (V _G ) and drain current (I _D ) characteristics are: Ag / AgCl reference electrode, saturated KCl (3.3 mol / l, Wako), and 3.3% agar salt containing KCl Measurement was performed at room temperature with a semiconductor parameter analyzer (B1500A, Agilent) under the condition of drain voltage (V _D ) = 2.5 V with the glass ring filled with ultrapure water using a bridge.

In this example, ultrapure water having a specific resistance of 18 MΩ · cm (manufactured using an ultrapure water production apparatus Urpure (Komatsu Electronics Co., Ltd.)) was used.

The sample magnetic beads prepared in (4) were washed once with ultrapure water immediately before the measurement, and the concentration was adjusted with ultrapure water to obtain a product concentration. After the adjustment, 4 μl of each solution containing the sample magnetic beads was gently added to 400 μl of ultrapure water so that the gate sensing part of the FET was covered with particles. About 5 minutes later, the amount of change in the gate voltage (V _G ) when the drain current (I _D ) = 1 mA before and after the addition of the particles was measured as the amount of change in the threshold voltage (ΔV _T ) caused by the addition of the sample magnetic beads. did. This measurement is performed on each sample magnetic bead, and the amount of change in threshold voltage (ΔV _T ) obtained is compared to determine the presence or absence of biotin reacted with the biotin magnetic particle-streptavidin complex. It was measured electrically as the change in charge density above.

The schematic diagram of this experiment is shown in FIG. 2, and the measurement results of the electrical characteristics of each sample magnetic bead are shown in FIG.

(5) Cleaning of FET After the measurement, the magnet placed under the gate of the ISFET was removed, and the sample particles on the gate were roughly removed by pipetting. Thereafter, the sample particles remaining on the FET gate surface were removed by ultrasonic cleaning with acetone, ethanol, and water for 1 minute each. Confirmation of particle removal was performed visually using an optical microscope (Keyence). When the removal of particles on the gate was confirmed, the FET was used to measure the next sample particle. When particles remained on the gate, ultrasonic cleaning was performed again.

Comparative Example 1: MCC method using biotin-streptavidin system (measured in buffer)

The electrical characteristics of the sample magnetic beads were measured in the same steps as in Example 1 except that the measurement was performed in a buffer solution (10 μM phosphate solution containing Na ₂ HPO ₄ and NaH ₂ PO ₄ (Wako), respectively). The experiment was conducted. The measurement results of the electrical characteristics of each sample magnetic bead are shown in FIG.

The results obtained in Example 1 and Comparative Example 1 are summarized in the following table.

As is apparent from Table 1, in the present invention, by measuring the electrical properties of the sample magnetic beads in pure water, surprisingly, compared with the measurement in a buffer solution which is a conventional method, The measurement limit concentration of the target substance decreased by 2 orders. Furthermore, also from the viewpoint of detection sensitivity, in the present invention, it is increased several times to several tens of times as compared with measurement in a buffer solution (for example, when compared at a target substance concentration of 0.9 fM, It can be seen that the detection sensitivity increased by 56.6 / 2.0≈about 30 times compared with the measurement in the buffer solution).

A technique for detecting a substance having an aM (atmolar: 10 ⁻¹⁸ M) level concentration with high detection sensitivity using a simple apparatus such as the present invention has not been known so far. In addition, by replacing the factor that binds to the magnetic beads and specifically binds to the target substance with a nucleic acid, an antibody, or the like, the present system can be used to detect various target substances. The ability to detect nucleic acids using the MCC method is disclosed in Japanese Patent Application Laid-Open No. 2012-80873 and Eur Biophys J (2014) 43: 217-225 by the present inventors. Example 2 below shows that the MCC method can also be used in an antigen-antibody reaction system.

Example 2: MCC method using an antigen-antibody reaction system

(1) Modification of the surface of magnetic beads with amino groups In this example, φ1 μm magnetic beads (7-12 × 10 ⁹ beads / ml, Dynabeads MyOne Carboxylic Acid, purchased from Invitrogen) having carboxylic acid groups on the surface were used. used. A 50 μl solution containing magnetic beads was dispersed in a microtube and washed twice with deionized water. The supernatant was removed while attracting the magnetic beads using a magnet. Diaminodecane (1,10-diaminothecode) in heated deionized water, containing 6 mM WSC (1-ethyl-3- (3-dimethyl-aminopropylol) carbodiimide hydrochloride, Dojindo Laboratories Co., Ltd.) , Ltd.) was dissolved at a saturated concentration to prepare a diaminodecane solution. The magnetic beads were dissolved in 100 ul of the diaminodecane solution and left at 50 ° C. overnight to modify the amino group on the surface of the magnetic beads. Then, the magnetic beads were collected by a magnet and washed with deionized water twice at room temperature to remove residual reagents.

The magnetic beads at this stage have one of the two amino groups of diaminodecane bonded to the carboxylic acid group on the surface of the magnetic beads, and the other amino group not used for bonding is free. .

(2) Immobilization of antibody on the surface of magnetic beads In this example, Monoclonal Anti-Dinitropheny produced Produced in mouse (IgE isotype, CHL Spel-7, Affinity purine ligine ligine ligine ligine ligine ligine ligine ligine immunized ligine immunized ligine infused in mouse) )It was used. Dispense 20 ul of the magnetic beads with amino groups on the surface prepared as described above into a microtube at a stock concentration, and wash twice with phosphate buffer saline (PBS pH 7.4 (1x), gibco) while attracting with a magnet. did. After discarding the supernatant, 20 ul of IgE fixing solution (60 mM WSC, PBS containing 0.1 μg / μl IgE (−)) was added, stirred by pipetting, and then incubated at 50 ° C. overnight. After the incubation, the magnetic beads were washed twice with PBS (−) at room temperature while being attracted with a magnet to wash away the unfixed IgE antibody and the residual reagent. Through the above treatment, the IgE antibody was immobilized on the surface of the magnetic beads.

In this example, the magnetic bead-IgE complex prepared here (hereinafter sometimes referred to as a sample magnetic bead in this example) is a “factor and a magnetic substance that specifically binds to a target substance”. Corresponding to “complex containing particles”.

(3) Antigen-antibody reaction on magnetic beads For antigen-antibody reaction, Albumin from Bovine Serum, 2,4-Dinitrophenylated (DNP-BSA, Invitrogen) was used as an antigen. The sample magnetic beads prepared in (2) were dispensed into microtubes at a stock concentration of 5 ul, the sample magnetic beads were collected using a magnet, and the supernatant was discarded. Thereafter, 20 ul of PBS (-) containing 0.1 mg / ml of the antigen was added, stirred by pipetting, and then incubated at room temperature for 30 minutes to carry out an antigen-antibody reaction. After the reaction, unreacted antigen was washed away by washing the sample magnetic beads twice with PBS (−) at room temperature while attracting the sample magnetic beads using a magnet.

(4) Measurement of antigen-antibody reaction by Molecular Charge Contact (MCC) method For the measurement of electric signal by MCC method, a commercially available N-channel depletion type Ion Sensitive FET chip (ISFET COM Co., Ltd.) that can confirm the Nernst response. Was used. As the gate structure of the FET chip, the insulating film has a laminated structure of Ta ₂ O ₅ / SiO ₂ , and the film thickness is about 120 nm in total. At the time of measurement, a glass ring (inner diameter: 10 mm, thickness: 1 mm) is attached with an epoxy resin so that the gate sensing part (10 × 340 μm) of the FET can be immersed in 400 μl of the measuring solution so that the sample magnetic particles are attracted to the sensing part. A magnet (neodymium, approximately 26 × 76 mm) was laid under the ISFET. The FET electrical characteristics such as gate voltage (V _G ) and drain current (I _D ) characteristics are: Ag / AgCl reference electrode, saturated KCl (3.3 mol / l, Wako), and 3.3% agar salt containing KCl Measurement was performed at room temperature with a semiconductor parameter analyzer (B1500A, Agilent) under the condition of drain voltage (V _D ) = 2.5 V with the glass ring filled with ultrapure water using a bridge.

In this example, ultrapure water having a specific resistance of 18 MΩ · cm (manufactured using an ultrapure water production apparatus Urpure (Komatsu Electronics Co., Ltd.)) was used.

The sample magnetic beads subjected to the antigen-antibody reaction in (3) were washed once with ultrapure water immediately before the measurement, and the concentration was adjusted with ultrapure water to obtain a product concentration (7-12 × 10 ⁹ beads / ml). After the adjustment, 4 μl of each solution containing the sample magnetic beads was gently added to 400 μl of ultrapure water so that the gate sensing part of the FET was covered with particles. About 5 minutes later, the amount of change in the gate voltage (V _G ) when the drain current (I _D ) = 1 mA before and after the addition of the particles was measured as the amount of change in the threshold voltage (ΔV _T ) caused by the addition of the sample magnetic beads. did. This measurement is performed on each sample magnetic bead, and by comparing the obtained threshold voltage change amount (ΔV _T ), the antigen-antibody reaction occurring on the sample magnetic bead is compared with the charge density on the gate surface. The change was measured electrically.

FIG. 5 shows an image diagram of the antigen-antibody reaction on the magnetic beads carried out in this example and the measurement results. As shown in FIG. 5, according to the present invention, sample magnetic beads in which an antigen-antibody reaction has occurred can be clearly distinguished from sample magnetic beads in which no antigen-antibody reaction has been performed. That is, it was shown that the MCC method can be applied to an antigen-antibody reaction system.

Claims (20)

A method for detecting the presence / absence of a target substance in a test sample using a field effect transistor,
(A) contacting a test sample with a complex containing a factor that specifically binds to the target substance and a magnetic particle in a solvent;
(B) after step (a), removing the solvent and dispersing the composite in water having a specific resistance of 1 MΩ · cm or more;
(C) after step (b), measuring the electrical characteristics of the composite with the field effect transistor while the composite is brought into close contact with the gate electrode surface of the field effect transistor by magnetic force; and
(D) The presence or absence of the target substance in the test sample by comparing the electrical characteristics measured in step (c) with those in a control experiment in which the complex is not contacted with the target substance. Detecting the absence,
Including,
Method. The method of claim 1, comprising:
The “water having a specific resistance of 1 MΩ · cm or more” is water having a specific resistance of 15 MΩ · cm or more,
Method. The method according to claim 1 or 2, comprising:
The “factor that specifically binds to the target substance” is an antibody that specifically binds to the target substance or an antigen-binding fragment thereof, or a nucleic acid that specifically binds to the target substance.
Method. The method according to claim 1 or 2, comprising:
The target substance is biotin, and the “factor that specifically binds to the target substance” is streptavidin,
Method. A method according to any one of claims 1 to 4, comprising
The gate surface of the field effect transistor is surface-treated so as to generate a surface functional group in a solution,
Method. 6. A method according to claim 5, wherein
The gate electrode surface of the field effect transistor is coated with oxide or nitride,
Method. The method of claim 6, comprising:
The oxide or nitride is tantalum oxide, aluminum oxide, silicon nitride, titanium oxide, or silicon oxide,
Method. 6. A method according to claim 5, wherein
When the surface of the gate electrode of the field effect transistor is coated with a metal that does not substantially generate a surface functional group in the solution, the surface treatment is further performed so that the surface functional group is generated on the surface of the gate electrode in the solution. It is characterized by being
Method. The method according to claim 8, comprising:
The metal is gold or platinum,
Method. A method according to any one of claims 1 to 9,
The field effect transistor is an Ion Sensitive FET,
Method. An apparatus for detecting the presence / absence of a target substance in a test sample,
Field effect transistors,
A complex comprising a factor that specifically binds to the target substance and magnetic particles, and
A magnetic force generation source for drawing the composite to the surface of the gate electrode of the field effect transistor;
The apparatus measures the electrical characteristics of the composite while bringing the composite into close contact with the surface of the gate electrode of the field effect transistor by magnetic force, and the measured electrical characteristics are contacted with the target substance. By detecting the presence / absence of the target substance in the test sample by comparing with the electrical characteristics in the control experiment when not
The measurement is performed in a state where the complex exists in water having a specific resistance of 1 MΩ · cm or more,
apparatus. The apparatus of claim 11, comprising:
The “water having a specific resistance of 1 MΩ · cm or more” is water having a specific resistance of 15 MΩ · cm or more,
apparatus. The apparatus according to claim 11 or 12, comprising:
The “factor that specifically binds to the target substance” is an antibody that specifically binds to the target substance or an antigen-binding fragment thereof, or a nucleic acid that specifically binds to the target substance.
apparatus. The apparatus according to claim 11 or 12, comprising:
The target substance is biotin, and the “factor that specifically binds to the target substance” is streptavidin,
apparatus. The apparatus according to any one of claims 11 to 14,
The gate surface of the field effect transistor is surface-treated so as to generate a surface functional group in a solution,
apparatus. The apparatus of claim 15, comprising:
The gate electrode surface of the field effect transistor is coated with oxide or nitride,
apparatus. The apparatus of claim 16, comprising:
The oxide or nitride is tantalum oxide, aluminum oxide, silicon nitride, titanium oxide, or silicon oxide,
apparatus. The apparatus of claim 15, comprising:
When the surface of the gate electrode of the field effect transistor is coated with a metal that does not substantially generate a surface functional group in the solution, the surface treatment is further performed so that the surface functional group is generated on the surface of the gate electrode in the solution. It is characterized by being
apparatus. The apparatus of claim 18, comprising:
The metal is gold or platinum,
apparatus. The apparatus according to any one of claims 11 to 19, comprising:
The field effect transistor is an Ion Sensitive FET,
apparatus.

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